A cloned segment (450 bp) of the African green monkey (Cercopithecus aethiops) genome that contains DNA sequences homologous to the control region of simian virus 40 is being studied. Cellular transcripts that go in both possible directions from within the SV40-like region, which is hypersensitive to DNase I in monkey chromatin, have been detected. Also, the sequence provides information for initiation of transcription from vectors constructed by molecular cloning. The SV40-line region also is a bidirectional transcriptional start site in vitro reactions using fractionated cell free extracts. In vitro transcription depends on the presence of a transcription factor (Sp1) that is also required for in vitro transcription from SV40 DNA itself but not for other host cell promoters tested. The Sp1 factor binds to the cloned monkey genomic region, as demonstrated by footprinting experiments using DNase I. The SV40 control region and the monkey segment compete for binding to Sp1. With both DNAs, the binding is to homologous regions containing multiple repeats of the sequence 5'-GGGCGGPuPu. The data suggest that there is a special class of cellular promoters that, like the SV40 promoter, depend on the presence of the short G-rich DNA segment and interaction with a specific factor, Sp1.